A practical asymmetric synthesis of enantiopure spiro[4,4]nonane-1,6-dione, a valuable precursor for chiral ligand development, is reported. This synthetic strategy includes a kinetic resolution of the readily synthesized ketone precursor with a chiral quaternary carbon center by bioreduction with baker's yeast as the key step, followed by a hydroformylation, oxidation, esterification and Dieckmann cyclization reaction sequence to generate the spiro five-membered ring. It was found that the masking of the beta-ketone carbonyl group of enantiopure ethyl 1-allyl-2-oxocyclopentanecarboxylate via formation of a ketal with 1,3-diol derivative is necessary during the process of Dieckmann condensation in order to prevent its racemization under basic conditions. This method allows the gram-scale preparation of both enantiomers of spiro[4,4]nonane-1,6-dione (1) with excellent enantiopurities (up to >99% ee) in the overall yields of 54% [(R)-1] and 42% [(S)-1], respectively. The practicality of the present synthetic procedure has provided a fundamental platform for the development of spiro[4,4]nonane-1,6-dione-based chiral chemistry.